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• W1964908508 abstract "To the Editor: HIV-1 is subdivided into groups M, N, and O. Group O was first identified in 1994. 1,2 Diagnosis and follow-up of infants born to mothers infected by HIV-1 group O have been problematic, owing to a lack of methods adapted to these variants. Recently, viral RNA quantification in plasma was made possible by the advent of the LCx HIV RNA QT kit (Abbott, Chicago, IL) and our own real-time polymerase chain reaction (PCR) method based on Lightcycler (LC) technology (Roche Diagnostics GmbH, Germany). 3 The lack of a commercial screening kit for HIV-1 group O proviral DNA means that infection cannot be confirmed or ruled out in children with undetectable plasma viral RNA, which is notably the case during antiretroviral prophylaxis. To improve the diagnosis and management of vertical HIV-1 group O transmission, we have developed a real-time PCR method on LC that specifically detects the proviral DNA of these variants and have also optimized our quantitative plasma RNA assay method. We used these 2 methods to diagnose and monitor 4 children born in France to mothers infected by HIV-1 group O. METHODS AND PATIENTS Peripheral blood mononuclear cells (PBMCs) and plasma were separated by Ficoll-Hypaque density gradient centrifugation and stored at −80°C. 4 DNA was extracted with the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Germany) as recommended by the manufacturer. The final elution volume was 50 μL. The amount of DNA in each extract was determined by real-time PCR specific for the albumin gene, 5 with human genomic DNA as standard (Roche Diagnostics). RNA was extracted using the Magna Pure LC device and the Total Nucleic Acid kit, according to the manufacturer’s instructions (Roche Diagnostics). The initial plasma volume was 200 μL and volume of eluate was 50 μL. PCR detection of proviral DNA and plasma RNA quantification by RT-PCR were based on the same primers P1 5′-CTCAATAAAGCTTGCCTTGA-3′ and P2 5′-CGCCACTGCTAGAGATTTT-3′ and the probe FAM-5′-AAGCAGTGTGTGCTCATCTGTTG-3′ -Eclipse Dark Quencher (Eurogentec, Seraing, Belgium). These primers amplify a fragment of 111 bp located in the long terminal repeat region of HIV-1 group M and O strains. Revelation is based on a group O–specific Double-Dye probe as previously published, 3 but to reduce background during revelation, the TAMRA quencher has been replaced by the Eclipse Dark quencher. Proviral DNA was PCR-amplified using the LC-Faststart DNA master hybridization probes kit. The LC master mix (2 μL) was mixed with 4 mM MgCl2, 0.5 μM each primer, and 0.5 μM probe. A variable volume of extract, containing 500 ng of DNA, was added, and the final volume was made up to 20 μL with water. Amplification was carried out as follows: 95°C for 10 minutes (1 cycle), denaturation at 95°C for 10 seconds, and annealing at 58°C for 30 seconds (45 cycles). A synthetic plasmid 3 diluted with human DNA to 2 concentrations (5000 and 50 copies/500 ng of DNA) served as the positive control for each run. To determine the sensitivity of the PCR method for proviral DNA, the plasmid standard was diluted in human DNA to 10, 5, and 1 copies/500 ng of DNA, and each concentration was tested 5 times. RT-PCR previously described in a 2-step procedure was simplified. 3 Onestep RT-PCR was performed with the LC-RNA Master Hybridization Probes kit (Roche Diagnostics) with 2.5 mM Mn(OAc)2, 0.5 μM each primer, and 0.5 μM probe. Ten microliters of RNA solution was added to 10 μL of this mixture. Amplification was carried out as follows: reverse transcription 61°C for 30 minutes, 95°C for 30 seconds (1 cycle), denaturation 95°C for 1 second, annealing 58°C for 5 seconds, elongation 65°C for 30 seconds, and reading 55°C for 3 seconds (45 cycles). The quantification standard was the supernatant of an HIV-1 group O clade A strain (YBF32) amplified by cell culture and quantified 4 times by LCx. The sensitivity of this new version of the assay was determined by testing samples containing 500, 200, and 100 copies/mL, 5 times each. We then tested clinical samples from the 4 infants (babies 1, 2, 3, and 4) and their mothers (mothers 1, 2, 3, and 4). The 4 mothers, all from Cameroon, were diagnosed with HIV-1 group O infection by means of specific peptide-based assays, as previously described, with confirmation by sequencing. 6,7 The mothers and babies were screened for proviral DNA just after delivery, and the infants were retested later to confirm the initial results. Plasma RNA was quantified in the same samples, in parallel, by means of optimized real-time RT-PCR, and by using the LCx HIV RNA QT kit according to the manufacturer’s procedures. RESULTS In the proviral DNA screening test, all the standards containing 10 and 5 copies/500 ng of DNA were positive, but only 40% of the samples containing 1 copy/500 ng of DNA were positive. The detection limit of the method was thus set at 5 copies/500 ng of DNA. The sensitivity of the plasma RNA quantification assay was reevaluated: all the standards containing 500 and 200 copies/mL were positive, but only 60% of standards containing 100 copies/mL were positive. The quantification limit was thus set at 200 copies/mL. Proviral DNA was detected in all the mothers, confirming that the PCR method amplified transmissible virions (Table 1). Babies 1, 2, and 3 were negative for proviral DNA, while baby 4 was positive. Good agreement on plasma viral load values was obtained with the LCx and LC real-time PCR methods (Table 1). Viral load was below the quantification cutoff of both techniques in mother 1, and was quantifiable in mothers 2 and 3, with a difference of <0.5 log between the 2 methods. Viral load in mother 4 was 4.4 log in the LCx technique and 4.6 log in the LC technique. Viral load assay confirmed the negativity of babies 1, 2, and 3, and confirmed that baby 4 was infected. PBMCs from baby 4 were then co-cultured with phytohemagglutinin-activated PBMCs from seronegative subjects. P24 antigen was detected in the culture supernatant, and proviral DNA was detected in end-cultured cells with our PCR method.TABLE 1: Follow-up of Mothers and BabiesDISCUSSION Our PCR developed for group O proviral DNA can be used to diagnose the infection in infants who receive anti-retroviral treatment from birth and who have undetectable viral load. Moreover, we have optimized and simplified the plasma RNA assay by including automated extraction, a one-step RT-PCR, and a new quencher. The standard range is now prepared from a culture supernatant, which is treated identically to clinical samples. The quantification limit is 200 copies/mL, representing a marked improvement over the previous assay (1500 copies/mL). 3 To date, 65 HIV-1 group O–infected patients resident in France have been identified, two-thirds of whom are women of child-bearing potential, and mother-to-child transmission of HIV-1 group O has been documented. 8 The precise risk of transmission is not known, owing to the small number of patients. In our study, none of the 3 children whose mothers were diagnosed sufficiently early in pregnancy and were treated at the end of pregnancy was infected. The serologic status of mother 4, whose baby was infected, was not known at the time of delivery, and she was therefore not treated. Her CD4 cell count was 78/mm3, pointing to high viral load. An-tiretroviral treatment started after delivery drove her viral load below the detection limit (Table 1). At age 6 months, the child developed rapidly progressive disease with early signs of HIV encephalopathy, profound immune deficiency (CD4 cell count 400/mm 3), and pulmonary interstitial lymphoid disease. Treatment with the zidovudine, lamivudine, and lopinavir/ritonavir combination was started on day 186 of life and drove viral load below the detection limit (data not shown). In conclusion, it is now possible to diagnose and monitor HIV-1 group O infection with specific serologic and molecular tools. Proviral DNA screening can be used to diagnose children born to infected mothers, and also patients with undetectable plasma viral RNA, as recently reported with LCx and LC technology. 3,9 ACKNOWLEDGMENTS The authors thank all the technicians of the Virology Unit. Marie Gueudin, PharmD* Véronique Lemée, PharmD* Virginie Ferre, PharmD, PhD† Agnès Beby-Defaux, MD, PhD‡ Jean-Paul Pathé, MD§ Odile Guist'hau, PharmD Joséphine Braun, PhD* François Simon, Pr, MD, PhD*,†† Jean-Christophe Plantier, PharmD, PhD* *Unité de Virologie, GRAM EA 2636, CHU Charles Nicolle, Rouen, France; †Unité de Virologie EA 1156, CHU Nantes, France; ‡Laboratoire de Virologie, CHU de Poitiers BP 577, France; §Service de Médecine Interne, Centre Hospitalier, Evreux, France; ¶Laboratoire de Virologie, CHU de Rennes, France; and ††Institut Pasteur de Dakar, Dakar, Sénégal." @default.
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• W1964908508 title "Virologic Diagnosis and Follow-up of Children Born to Mothers Infected by HIV-1 Group O" @default.
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